We propose the development of a platform to accelerate the discovery of synthetic lethal interactions in human cancers. This work makes use of a human genome-wide library of lentiviral RNAi constructs recently developed by our lab and others (collectively termed "The RNAi Consortium" (TRC)) that targets over 18,000 human genes using over 90,000 lentiviral clones, yields stable suppression of target genes, and is compatible with both dividing and non-dividing cells. To enable miniaturized, high-throughput, arrayed loss- of-function screens with this library, our first aim will be to optimize the properties of lentivirus-infected cell microarrays (LICMs), a technology currently under development in our laboratory. Specifically, this aim will focus on optimizing the number of viruses deposited on each microarray feature by increasing the density of functional groups on the array surface using nanostructured surface features and/or biocompatible hydrogel coatings. In our second aim, we will fabricate whole-genome LICMs and demonstrate their feasibility toward large-scale loss-of-function screens. In our final aim, we will apply these systems to a screen that will be used to identify synthetic lethal interactions in cancers harboring deficiencies in PTEN, a tumor suppressor gene that is frequently implicated in human cancers. Specifically, in this aim we will use an engineered U-87 human cell line with tetracycline inducible PTEN expression to first identify genes that are synthetic lethal with PTEN, then test these candidate genes on a panel of human cancer cell lines with impaired PTEN function to identify those genes which are synthetic lethal in a range of diverse genetic and physiological contexts. The long-term objectives of this project are: (1) to identify genes which are synthetic lethal with PTEN, and thus targets for cancer drug development, and (2) to establish a robust, broadly useful arrayed screening platform to accelerate progress in functional genomics. Lay description: We propose to develop a high-throughput screening platform which will allow for the rapid and systematic identification of new, selective drug targets for human cancers. This project involves three components: the optimization of the proposed screening platform;the application of this platform to genome-wide screening experiments;and the use of this platform to systematically identify new cancer drug targets. The screening platform developed in this study has the potential to become an enabling tool for high-throughput screening experiments throughout the biomedical research community, and the particular application of these systems to cancer may significantly accelerate the discovery of new cancer drugs.